I. PCR-Based DNA Detection Technologies
Nucleic acid detection assays and sensitivity. Known DNA and/or RNA detection techniques are based on the principle of complementarity. For example, an oligonucleotide sequence is selected based on its ability to form a complementary duplex with a desired or predetermined nucleic acid target sequence, and the complementary duplex is detected, indicating the presence of the targeted nucleic acid in the reaction mixture. Such hybridization based detection assays should, at least in principal, detect the nucleic acid of interest regardless of its concentration in the test sample. However, the sensitivity of such direct detection hybridization assays is limited, and although some highly sensitive technologies for direct nucleic acid detection are currently under development, amplification of targeted nucleic acids is an important component of typical DNA detection systems. Numerous amplification technologies are known in the art, the most notorious examples including: Strand Displacement Amplification (SDA) (Walker G. T. et al, U.S. Pat. No. 5,270,184; Dattagupta N. et al, U.S. Pat. No. 6,214,587; Walker G. T. et al (1996) Nucleic Acids Res., 24, 384-353); Rolling Circle Amplification (RCA) (Lizardi P., U.S. Pat. No. 5,854,033); Linear target Isothermal Multimerization and Amplification (LIMA) (Hafner G. J. et al (2001) BioTechniques, 30, 852-867); Loop-Mediated Amplification (LMA) (Notomi T. and Hase T., U.S. Pat. No. 6,410,278; Notomi T. et al (2000) Nucleic Acids Res., 28, e63); Isothermal Amplification using chimeric or composite RNA/DNA primers (Cleuziat P. and Mandrand B., U.S. Pat. No. 5,824,517; Kurn N. (2001) U.S. Pat. No. 6,251,639); Nucleic Acid Sequence-Based Amplification (NASBA) (Oehlenschlager F. et al (1996) Proc. Natl. Acad. Sci. USA, 93, 12811-12816; Davey C. and Malek L. T., U.S. Pat. No. 6,063,603); and other methods. By far, the most common element among these technologies is the use of oligonucleotide primers that form complementary hybridization complexes with desired/predetermined target sequences of the test nucleic acids, initiating synthesis of DNA copies and providing for target nucleic acid amplification.
Demands for quantitative measurements of nucleic acids of interest, for example, of limiting viral or bacterial loads in test samples, prompted development of real time assays in which the amplification products are detected as the amplification reaction progresses. This significantly decreases the number of subsequent handling steps required for the detection of amplified material and also helps to resolve amplification cross-contamination issue. Fluorescence can be detected at a nanomolar level well within the sensitivity and productivity of many known nucleic acid amplification technologies. Development of real time fluorescence-based detection assays has been reported for many nucleic acid amplification schemes, for example, a fluorescent probe called a Molecular Zipper was effectively combined with Rolling Circle amplification (Yi J. et al (2006) Nucleic Acids Res., 34: e81) and several fluorescence-based detection approaches were combined with NASBA amplification (Niesters H. G. (2001) Methods, 25: 419-429).
Polymerase chain reaction (PCR) methodology has revolutionized the detection of nucleic acids, where at least in theory, as little as a single copy of DNA or RNA can be amplified and detected. A typical PCR-based detection assay consists of at least two primers and a fluorescent probe. Fluorescence can be detected at the nanomolar level, which is well within the range of PCR sensitivity and productivity. PCR primers are typically designed to bind to opposite DNA strands; that is, the primers bind in an orientation such that extension of one creates a template for the other primer. The PCR reaction runs in cycles in which DNA fragments synthesized in the previous cycle are ‘strand-separated’ in a denaturation step (typically at 95° C.), followed by rapid cooling to start an ‘annealing-extension’ stage (typically carried out at 55-65° C.). In annealing stage, the primers bind again to the amplified strands and get extended by a thermophilic DNA polymerase. Under optimal PCR conditions, the concentration of the amplified DNA fragment doubles at each PCR cycle reaching a detectable level after ˜20-40 cycles depending on the initial target amount/load.
Application of fluorescence based methods led to development of many real time detection techniques (Clementi M. et al (1993) PCR Methods Appl. 2:191-196; Clegg R. M. (1992) Methods Enzymol., 211: 353-388; Freeman W. M. et al (1999) Biotechniques, 26: 112-122, 124-125; Didenko V. V. (2001) BioTechniques, 31: 1106-1121; Mackay I. M. et al (2002) Nucleic Acids Res., 30: 1292-1305; Mackay J., Landt O. (2007) Methods Mol. Biol., 353: 237-262; Higuchi R. et al (1992) Biotechnology, 10: 413-417; Higuchi R. et al (1993) Biotechnology, 11: 1026-1030; Lewin S. R. et al (1999) J. Virol., 73: 6099-6103; etc.). This significantly simplified nucleic acid detection eliminating the variability traditionally associated with quantitative PCR. A simple approach to detect DNA in PCR is based on use of fluorescent agents like ethidium bromide, YO-PRO-1, SYBR Green and other dyes which change their fluorescence properties upon the interaction with double stranded nucleic acids providing detectable signal (Ishiguro T. et al (1995) Anal. Biochem., 229: 207-213; Tseng S. Y. et al (1997) Anal. Biochem., 245: 207-212; Morrison T. B. et al (1998) Biotechniques, 24: 954-958; Schneeberger C. et al (1995) PCR Methods Appl., 4: 234-238; and Mackay J., Landt O. (2007) Methods Mol. Biol., 353: 237-262). The principal drawback to this method of DNA detection is that both specific and nonspecific products generate signal and this may lead to false positive results limiting applicability of the approach, in particular, in clinical diagnostics. Real-time systems were improved by probe-based PCR product detection. Fluorescent probes are oligonucleotides which typically labeled with two fluorescent dyes. The probes are designed to bind exclusively to a target amplicon usually between the primer binding sites providing the desired assay specificity. This also enables multiplex PCR wherein multiple target nucleic acids are simultaneously amplified and detected. The probe-based detection of PCR amplicon commonly employs Förster Resonance Energy Transfer (FRET) effects.
FRET-based detection. FRET is a distance-dependent interaction occurring between two dye molecules in which excitation is transferred from a donor to an acceptor fluorophore through dipole-dipole interactions without the emission of a photon. As a result, the donor molecule fluorescence is quenched, and the acceptor molecule becomes excited. The efficiency of FRET depends on spectral properties, relative orientation and distance between the donor and acceptor chromophores (Förster T. (1965) Delocalized excitation and excitation transfer. In Sinanoglu, O. (ed.), Modern Quantum Chemistry, Istanbul Lectures, part III. Academic Press, New York: 93-137). In the case of random dipole orientation and with a good overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, the efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation (Clegg R. M. (1992) Methods Enzymol., 211: 353-388; Clegg R. M. (1995) Curr. Opin. Biotech., 6: 103-110; Selvin P. R. (1995) Methods Enzymol., 246: 300-334), making FRET useful over distances comparable to the dimensions of biological macromolecules (Stryer L. and Haugland R. P. (1967) Proc. Natl. Acad. Sci. USA, 58:719-726). FRET is widely used in biomedical research and particularly in probe designs for nucleic acid detection (Didenko V. V. (2001) BioTechniques, 31, 1106-1121).
The acceptor chromophore may be a non-fluorescent dye chosen to quench fluorescence of the reporting fluorophore (Eftink M. R. (1991) In Lakowicz J. R. (ed.), Topics in Fluorescence Spectroscopy. Plenum Press, New York, V.2: 53-126). Formation of sequence-specific hybrids between the target nucleic acid and the probes leads to a changes in fluorescent properties of the probe providing for detection of the nucleic acid target. The real-time FRET based assays are particularly well suited for clinical diagnostics, because unlike the case of intercalating dyes and fluorescent agents (e.g. ethidium bromide, SYBR Green) discussed above, the detection is sequence-specific, virtually eliminating false positive results.
‘Hybridization-triggered’ FRET probes. Many detection strategies and designs exploiting the FRET effect are known in the art. One strategy is a hybridization-triggered FRET probe approach, based on distance change between the donor and acceptor dyes as result of a sequence specific complex formation between a target nucleic acid and a fluorescent oligonucleotide probe. For example, the ‘Adjacent Hybridization Probe’ method utilizes two oligonucleotide probes hybridizing to adjacent target DNA sequences as described in e.g. Eftink M. R. (1991) In Lakowicz J. R. (ed.), Topics in Fluorescence Spectroscopy. Plenum Press, New York, V.2 53-126; Heller M. J. and Morrison L. E. (1985) In Kingsbury, D. T. and Falkow, S. (eds.), Rapid Detection and Identification of Infectious Agents. Academic Press, New York, 245-256; Cardullo R. A. et al (1988) Proc. Natl. Acad. Sci. USA, 85:8790-8794. Each of the probes is labeled by one of FRET-pair dyes at an appropriate probe end such that when both probes are hybridized to a target DNA, the donor and acceptor fluorophores are brought in spatial proximity of each other providing for a detectable FRET interaction.
‘Self-Quenching Fluorescence’ probes. An alternative approach is the use of ‘Self-Quenching Fluorescence’ probes described in Livak K. J. et al, U.S. Pat. No. 5,723,591. These probes include fluorescent reporter and quencher moieties conjugated to opposite ends of the same probe. Due to random oligonucleotide coiling, the quencher moiety is sufficiently close to the reporter dye to quench its fluorescence. However, once the probe is hybridized to a complementary polynucleotide target, the quencher and reporter moieties are separated, thus enabling the reporter dye to fluoresce. The Self-Quenching Fluorescence probes approach has been limited because of an inefficient FRET effect in the unhybridized probe that leads to an elevated fluorescence background. The background problem can be resolved by synthesizing the oligonucleotide with a flexible PNA backbone (e.g. Ortiz E. et al (1998) Mol. Cell. Probes, 12, 219-226).
‘Molecular beacon’ probes. Efficient FRET is achieved in ‘Molecular Beacons’, which are hairpin-shaped oligonucleotide probes in which the FRET dyes are brought in close proximity by intramolecular stem formation (e.g. Tyagi S, and Kramer F. R. (1996) Nat. Biotechnol., 14: 303-308; Bonnet G. et al (1999) Proc. Natl. Acad. Sci. USA, 96:6171-6176; Tyagi S. et al (2000) Nat. Biotechnol., 18:1191-1196; Marras S. A. E. et al (2002) Nucleic Acids Res., 30 e122). Hybridization of a Molecular Beacon probe to a complementary polynucleotide target opens the hairpin probe structure and separates the quencher and reporter moieties enabling the reporter dye to fluoresce. Molecular Beacons are known for their remarkably low fluorescence background, and these probes are well adapted for use in real-time PCR as described in, e.g. Piatek A. S. et al (1998) Nat. Biotechnol., 16:359-363; Lewin S. R. et al (1999) J. Virol., 73 6099-6103. Molecular Beacons have also improved polymorphism discriminating capabilities.
‘Scorpion’ primers. Covalent linking of a ‘molecular beacon’ probe to one of the PCR primers is a unique property of ‘Scorpion’ primers, e.g. Whitcombe D. et al (1999) Nature Biotech., 17:804-807; Thelwell N. et al (2000) Nucleic Acids Res., 28:3752-3761. In Scorpions the 5′-end of a PCR primer is conjugated to the 3′-end of a molecular beacon through a long, flexible linker. The linker is not a template for DNA polymerase, thus precluding extension over the beacon sequence. The genomic part of the molecular beacon is designed to bind to a targeted extension product of the primer to which the probe is covalently linked. Unlike Molecular Beacons, the DNA detection stage in Scorpions comprises an intra-molecular reaction. This property helps to overcome a known problem of the Beacon technology associated with slow kinetics of hybridization.
Eclipse probes. Eclipse probes are yet another example of hybridization-based FRET probes that have low fluorescence background (Afonina I. A. et al (2002) BioTechniques, 32 940-949). The Eclipse probe design includes a minor groove binding (MGB) moiety at the 5′-end in addition to two FRET dyes one of which is a non-fluorescent or dark quencher. Due to the strong, DNA-duplex stabilizing effect of the MGB-moiety (Kutyavin I. V. et al (1997) Nucleic Acids Res., 25:3718-3723), the probes can be designed to be as short as 12-20-mers and yet maintain the hybridization properties required for real-time PCR detection. Placing the MGB-tail at 5′-end of the probes completely blocks 5′-nuclease cleavage and the fluorescent signal is generated solely due to the hybridization-triggered dye separation. Fluorescence background is low and Eclipse probes readily discriminate SNPs.
‘Self-Quenched Fluorogenic primers’ or ‘LUX’ primers. The mechanism of FRET disruption by the distancing of FRET dyes poses certain limits. For example, it is difficult to completely abolish the FRET effect, and probes have to be at least 20-24-mers. In short 8-12 by probe-target duplexes, “residual” quenching can reach as high as 20-50% (Cardullo R. A. et al (1988) Proc. Natl. Acad. Sci. USA, 85:8790-8794). Furthermore, the reporter dye can be partially quenched by neighboring bases, in particular, by guanines regardless of the limited spectral overlap. This effect is well known and has been used in a DNA detection technology known by the name of ‘Self-Quenched Fluorogenic primers’ or ‘Light Upon eXtension’ (LUX primers), e.g. Nazarenko I. et al (2002) Nucleic Acids Res., 30: e37; Nazarenko I. et al (2002) Nucleic Acids Res., 30: 2089-2195. The technology performs best with “green” dyes like fluorescein (FAM). However, LUX primers are not sequence-specific. Any product of a LUX primer extension, including primer-dimers, will generate a fluorescent signal.
Cleavable dual labeled FRET probes; TaqMan™. The best strategy to abolish FRET is based on cleavage of the oligonucleotide probes upon their binding to target nucleic acids. TaqMan™ technology was developed as a real-time nucleic acid detection method and utilizes the 5′-3′ exonuclease activity of Thermus aquaticus (Taq) polymerase, e.g. Lie Y. S. and Petropoulos C. J. (1998) Curr. Opin. Biotech., 9:43-48. A dual labeled FRET probe is designed to anneal to a target sequence located between two PCR primer binding sites. During strand elongation, Taq polymerase cleaves the probe that is hybridized down stream from a primer site releasing the reporter dye from the quencher and thus permanently and irreversibly disrupting FRET, e.g. Livak K. J. et al (1995) PCR Methods and Applications, 4:357-362. Since the probe cleavage is irreversible, the signal generated at a given PCR cycle is a sum of signals generated at that particular cycle plus all previous ones. However, elevated fluorescence background of the “classical” TaqMan™ probes tends to overshadow this advantage. Conjugation with an MGB-moiety at the 3′-end leads to significant improvement of this parameter (Kutyavin I. V. et al (2000) Nucleic Acids Res., 28:655-661). Relatively short 12-18-mer MGB-TaqMan™ probes have improved SNP discriminating properties. However, TaqMan™ technology is still tightly bound to PCR performance whereas Cycling Probe Technologies (CPT) are relatively independent.
Cycling Probe Technologies (CPT). Cycling Probe Technologies (CPT) are also preferred detection systems for practicing methods of the invention. These reactions are based on continuous cleavage of oligonucleotide probes which bind to a target nucleic acid in a sequence-specific fashion. An appropriate endonuclease recognizes the complex and cleaves the probe while leaving the target strand intact recycling it for the next round of cleavage. If the hybridized probe is cleaved internally, the cleavage products form weaker hybrids than the original probe and these probe fragments dissociate from the target strand leaving it available for additional rounds of the cleavage reaction. Target recycling means that more than one probe can be cleaved per target molecule. Unlike all other technologies referred above, including TaqMan™, in CPT reactions the signal is a function of two main variables, target concentration and time. When the target concentration is fixed, the signal grows linearly in time. Reflecting the reaction progress, cleavage slows down and eventually stops when essentially all CPT probes get cleaved. Several system designs have been reported. The first approach is based on use of chimeric DNA-RNA probes that are cleaved by RNAse H upon the binding to target DNA, as described in Fong W. et al (2000) J. Clin. Microbiol., 38: 2525-2529; Modruzan Z. et al (2000) Diagn. Microbiol. Infect. Dis., 37:45-50. These DNA probes are designed to have at least 4-5 ribonucleotides in the middle of the oligonucleotide chain. RNAse H cleaves only the RNA portion of the hybridized probe and the target polynucleotide is recycled to hybridize to another uncleaved probe molecule. Under appropriate conditions, this leads to a cycling of the probe cleavage reaction. Recent discovery and isolation of thermo-stable analogs of RNAse H have allowed combining this DNA detection technology with PCR as demonstrated in e.g. Harvey J. J. et al (2004) Anal. Biochem., 333: 246-255. The respective FRET probes may be obtained from Takara Bio.
The second CPT approach is based on the substrate specificity of Endonuclease IV from E. coli, an AP endonuclease that initiates repair of abasic sites and other related lesions in DNA. A FRET probe and enhancer can collectively form a substrate for the AP endonuclease that simulates a partially degraded abasic site. The enzyme recognizes this artificial substrate and “clips” the 3′-tail of the probe thereby releasing the reporter dye and disrupting FRET. This reaction can be performed in a cycling mode where a high yield of cleaved probe is achieved at nanomolar or even sub-nanomolar target DNA concentrations as described in Kutyavin I. V. et al (2004) US Patent Application #20040101893.
Third, and perhaps, the most advanced cycling probe technology on the market is the INVADER™ detection assay. It utilizes the flap or 5′-endonuclease activity of certain polymerases to cleave two partially overlapping oligonucleotides upon their binding to target DNA. The INVADER™ assay typically consists of two consecutive cycling cleavage reactions. The enzyme used to provide the cleavage reaction is CLEAVASE, a DNA polymerase with substantially reduced or completely eliminated synthetic capabilities, e.g. Dahlberg J. E. et al (1997) U.S. Pat. No. 5,691,142; Dahlberg J. E. et al (1998) U.S. Pat. No. 5,837,450; Brow M. A. D. et al (1998) U.S. Pat. No. 5,846,717; Prudent J. R. et al (1999) U.S. Pat. No. 5,985,557; Hall J. G. et al (1999) U.S. Pat. No. 5,994,069; Brow M. A. D. et al (1999) U.S. Pat. No. 6,001,567; Prudent J. R. et al (2000) U.S. Pat. No. 6,090,543; Prudent J. R. et al (2002) U.S. Pat. No. 6,348,314; Prudent J. R. et al (2005) U.S. Pat. No. 6,875,572; Aizenstein B. D. et al (2005) U.S. Pat. No. 6,913,881; Schweitzer B. and Kingsmore S. (2001) Curr. Opin. Biotech., 12: 21-27. The detection system is a very efficient signal amplification assay which may not require any prior target DNA amplification. However, prior amplification of nucleic acids is a preferred approach in applying the INVADER assay. The primary concern is background fluorescence that increases linearly with time. It is generated by non-specific cleavage of the cassette probe. Furthermore the assay requires substantial target DNA load, e.g. Schweitzer B. and Kingsmore S. (2001) Curr. Opin. Biotech., 12:21-27, when the amplification is not applied. Combinations of CPT with nucleic acid amplification techniques provide critical advantages as described for the oligonucleotide probes with secondary structures in Sorge J. A. (2001) U.S. Pat. No. 6,589,743.
Sorge J. A. also reported methods of nucleic acid detection wherein a cleavage structure comprising duplex and single-stranded nucleic acid is formed by incubating a sample comprising a target nucleic acid sequence with a probe having a secondary structure that changes upon binding to the target nucleic acid sequence, and cleaving said cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal. In certain embodiments, the hybridized probes are cleaved during PCR. Unlike the TaqMan™ assays, it has also been proposed to provide DNA polymerase and 5′-nuclease (FEN) activities which are necessary to perform the methods using different enzymes. Sorge J. A. also describes an approach of sequential cleavage reaction, similar to the ‘Invader’ assay, to enhance the signal detection reaction. The technology is collectively disclosed in many published patents, e.g. Sorge J. A. U.S. Pat. No. 6,350,580, U.S. Pat. No. 6,528,254, U.S. Pat. No. 6,548,250, U.S. Pat. No. 6,893,819, U.S. Pat. No. 7,118,860 and U.S. Pat. No. 7,183,052.
The cited and above-described nucleic acid detection technologies represent an exemplary fraction of innovations and methods in this field of art. There are many other techniques that are based on use of oligonucleotide probes and, in particular, FRET probes. All of the technologies that are based on hybridization of an oligonucleotide probe with a target nucleic acid would benefit from use the present invention. Detailed guidance in performing a particular detection reaction including the rules for oligonucleotide primer and probe designs, preferred composition of the reaction mixtures, reaction conditions, characteristics of the assays and its applicability and limitations, and other important information to carry out the detection reactions can be found in cited above manuscripts and patents which are incorporated herein by reference.
Regardless of the significant progress made to date, methods of nucleic acids detection are still not optimal. Therefore, there is a pronounced need in the art for versatile, effective but simple and inexpensive approaches providing fast and robust amplification and detection of target nucleic acids.